Short-Term Treadmill Running as a Model for Studying Cell-Free DNA Kinetics In Vivo

BACKGROUND Increased plasma concentrations of cell-free DNA (cf-DNA) are considered a hallmark of various clinical conditions. Despite intensive research in this field, limited data are available concerning the time course of release and clearance of cf-DNA in vivo. METHODS We extracted cf-DNA from plasma samples taken before and immediately after a 10-km cross-country run, and from samples taken before, immediately after, and 30 min after exhaustive short-term treadmill exercise. The contribution of nuclear (nDNA) and mitochondrial DNA (mtDNA) was measured by quantitative real-time PCR. The incremental treadmill exercise setup was exploited to delineate the precise sequencing and timing of cf-nDNA, lactate, and high-mobility group box 1 protein (HMGB1) release during the exercise and recovery phases. RESULTS Postexercise plasma cf-nDNA concentrations in cross-country and treadmill runners were significantly increased, by 7.6-fold and 9.9-fold, respectively (P < 0.001). cf-nDNA concentrations were not correlated with age, sex, or body mass index. Plasma concentrations of cf-nDNA and HMGB1 in postexercise samples of treadmill runners were significantly correlated (r = 0.84; P = 0.004). cf-mtDNA concentrations were not affected by treadmill exercise. Time-course analyses demonstrated that cf-nDNA is released within minutes after the onset of exercise and is rapidly cleared from the circulation after the cessation of exercise. Nearly congruent kinetics for cf-nDNA, lactate, and HMGB1 were observed during the exercise phase. CONCLUSIONS A single bout of exhaustive short-term treadmill exercise constitutes a versatile model system suitable for addressing basic questions about cf-DNA biology.

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Anti-viral properties of interferon beta treatment in patients with multiple sclerosis

Multiple Sclerosis Journal ◽

10.1191/1352458502ms794oa ◽

2002 ◽

Vol 8 (3) ◽

pp. 237-242 ◽

Cited By ~ 29

Author(s):

J Hong ◽

M V Tejada-Simon ◽

V M Rivera ◽

Y CQ Zang ◽

J Z Zhang

Keyword(s):

Multiple Sclerosis ◽

Treatment Efficacy ◽

Interferon Beta ◽

Viral Infections ◽

Human Herpesvirus ◽

Virion Protein ◽

Cell Free Dna ◽

Free Dna

Viral infections are potentially associated with the etiology and pathogenesis of multiple sclerosis (MS). It has been speculated that the treatment efficacy of interferon beta (IFN beta) in MS may relate to its anti-viral properties. The study was undertaken to evaluate the in vivo anti-viral effects of IFN beta-1a in patients with MS. Human herpesvirus-6 (HHV-6) was studied as an example for being a latent neurotropic virus. IFN beta used at concentrations of approximately 0.5 mg/ml was shown to significantly reduce in vitro HHV-6 replication in a susceptible T-cell line. Sera derived from 23 MS patients treated with IFN beta-1a were examined for serum cell-free DNA of HHV-6 as an indicator for viral replication and the reactivity of IgM antibodies to a recombinant HHV-6 virion protein containing a known immunoreactive region. The results were compared with those of control sera obtained from untreated MS (n=29) and healthy individuals (n=21). The findings indicated that IFN beta treatment significantly reduced HHV-6 replication as evident by decreased cell-free DNA in treated MS specimens. The results correlated with decreased IgM reactivity to the HHV-6 antigen in treated MS patients compared to untreated controls, suggesting reduced exposure to HHV-6. The findings were confirmed in paired sera obtained from seven MS patients before and after the treatment. The study provides new evidence indicating that IFN beta has potent in vivo anti-viral effects that may contribute to the treatment efficacy in MS.

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Neutrophil Extracellular Traps May Contribute to the Pathogenesis in Adult-onset Still Disease

The Journal of Rheumatology ◽

10.3899/jrheum.181058 ◽

2019 ◽

Vol 46 (12) ◽

pp. 1560-1569 ◽

Cited By ~ 4

Author(s):

Mi-Hyun Ahn ◽

Jae Ho Han ◽

Young-Jun Chwae ◽

Ju-Yang Jung ◽

Chang-Hee Suh ◽

...

Keyword(s):

Immunohistochemical Analysis ◽

Serum Levels ◽

Neutrophil Extracellular Traps ◽

Polymorphonuclear Neutrophils ◽

Adult Onset ◽

Cell Free Dna ◽

Extracellular Traps ◽

Free Dna ◽

Still Disease

Objective.Release of neutrophil extracellular traps (NET) has been described as an effector mechanism of polymorphonuclear neutrophils in several inflammatory diseases. Thus, this study was performed to evaluate the role of NET in the pathogenesis of adult-onset Still disease (AOSD).Methods.We determined the serum levels of NET molecules and investigated their associations with clinical disease activities in patients with AOSD. Further, we analyzed the differences in the NETosis response in AOSD patients compared to healthy controls (HC). To explore the in vivo involvement of NET in AOSD, we performed immunohistochemical analysis of skin and lymph node (LN) biopsies for proteins related to NET in patients with active AOSD.Results.Serum levels of cell-free DNA, myeloperoxidase (MPO)-DNA complex, and α-defensin were significantly increased in patients with AOSD compared to HC. Serum levels of the NET molecules, cell-free DNA, MPO-DNA, and α-defensin were correlated with several disease activity markers for AOSD. In followup of patients with AOSD after treatment with corticosteroid, the levels of cell-free DNA and α-defensin decreased significantly. On immunohistochemistry, neutrophil elastase–positive and MPO-positive inflammatory cells were detected in skin and LN of patients with AOSD, and were expressed in fiber form in the lesions. The serum from patients with active AOSD induced NETosis in neutrophils from HC. NET molecules induced interleukin 1β production in monocytes, representing a novel mechanism in the pathogenesis of AOSD.Conclusion.The findings presented here suggest that NET may contribute to the inflammatory response and pathogenesis in AOSD.

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Short-term exercise improves myocardial tolerance to in vivo ischemia-reperfusion in the rat

Journal of Applied Physiology ◽

10.1152/jappl.2001.91.5.2205 ◽

2001 ◽

Vol 91 (5) ◽

pp. 2205-2212 ◽

Cited By ~ 120

Author(s):

Haydar A. Demirel ◽

Scott K. Powers ◽

Murat A. Zergeroglu ◽

R. Andrew Shanely ◽

Karyn Hamilton ◽

...

Keyword(s):

Heat Stress ◽

Maximum Rate ◽

Treadmill Exercise ◽

Ischemia Reperfusion ◽

Left Ventricular ◽

Antioxidant Defenses ◽

Whole Body ◽

Sprague Dawley ◽

Short Term

These experiments examined the independent effects of short-term exercise and heat stress on myocardial responses during in vivo ischemia-reperfusion (I/R). Female Sprague-Dawley rats (4 mo old) were randomly assigned to one of four experimental groups: 1) control, 2) 3 consecutive days of treadmill exercise [60 min/day at 60–70% maximal O2 uptake (V˙o 2 max)], 3) 5 consecutive days of treadmill exercise (60 min/day at 60–70%V˙o 2 max), and 4) whole body heat stress (15 min at 42°C). Twenty-four hours after heat stress or exercise, animals were anesthetized and mechanically ventilated, and the chest was opened by thoracotomy. Coronary occlusion was maintained for 30-min followed by a 30-min period of reperfusion. Compared with control, both heat-stressed animals and exercised animals (3 and 5 days) maintained higher ( P < 0.05) left ventricular developed pressure (LVDP), maximum rate of left venticular pressure development (+dP/d t), and maximum rate of left ventricular pressure decline (−dP/d t) at all measurement periods during both ischemia and reperfusion. No differences existed between heat-stressed and exercise groups in LVDP, +dP/d t, and −dP/d t at any time during ischemia or reperfusion. Both heat stress and exercise resulted in an increase ( P < 0.05) in the relative levels of left ventricular heat shock protein 72 (HSP72). Furthermore, exercise (3 and 5 days) increased ( P < 0.05) myocardial glutathione levels and manganese superoxide dismutase activity. These data indicate that 3–5 consecutive days of exercise improves myocardial contractile performance during in vivo I/R and that this exercise-induced myocardial protection is associated with an increase in both myocardial HSP72 and cardiac antioxidant defenses.

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Circulating cell-free DNA is elevated in community-acquired bacterial pneumonia and predicts short-term outcome

Journal of Infection ◽

10.1016/j.jinf.2016.07.011 ◽

2016 ◽

Vol 73 (4) ◽

pp. 383-386 ◽

Cited By ~ 4

Author(s):

William W. Siljan ◽

Jan C. Holter ◽

Ståle H. Nymo ◽

Camilla Schjalm ◽

Fredrik Müller ◽

...

Keyword(s):

Bacterial Pneumonia ◽

Short Term ◽

Term Outcome ◽

Cell Free Dna ◽

Free Dna ◽

Short Term Outcome ◽

Circulating Cell Free Dna

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Neutrophil-Extracellular Traps, Cell-Free DNA, and Immunothrombosis in Companion Animals: A Review

Veterinary Pathology ◽

10.1177/0300985819861721 ◽

2019 ◽

Vol 57 (1) ◽

pp. 6-23 ◽

Cited By ~ 4

Author(s):

Robert Goggs ◽

Unity Jeffery ◽

Dana N. LeVine ◽

Ronald H. L. Li

Keyword(s):

Companion Animals ◽

High Mobility ◽

Extracellular Dna ◽

Death Process ◽

Cell Free Dna ◽

Pathogen Invasion ◽

Extracellular Traps ◽

Trap Formation ◽

Free Dna ◽

Extracellular Trap

Immunothrombosis is a potentially beneficial physiological process that aids innate immunity and host defense against pathogen invasion. However, this process can also be damaging when it occurs to excess or in critical blood vessels. Formation of extracellular traps by leukocytes, particularly neutrophils, is central to our understanding of immunothrombosis. In addition to degranulation and phagocytosis, extracellular traps are the third mechanism by which neutrophils combat potential pathogens. These traps consist of extracellular DNA decorated with bactericidal cellular proteins, including elastase, myeloperoxidase, and cathepsins. Neutrophils can release these structures as part of a controlled cell-death process or via a process termed vital NETosis that enables the cells to extrude DNA but remain viable. There is accumulating evidence that NETosis occurs in companion animals, including dogs, horses, and cats, and that it actively contributes to pathogenesis. Numerous studies have been published detailing various methods for identification and quantification of extracellular trap formation, including cell-free DNA, measurements of histones and proteins such as high-mobility group box–1, and techniques involving microscopy and flow cytometry. Here, we outline the present understanding of these phenomena and the mechanisms of extracellular trap formation. We critically review the data regarding measurement of NETosis in companion animals, summarize the existing literature on NETosis in veterinary species, and speculate on what therapeutic options these insights might present to clinicians in the future.

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Short-term alpha- or gamma-delta-enriched tocopherol oil supplementation differentially affects the expression of proinflammatory mediators: selective impacts on characteristics of protein tyrosine nitration in vivo

Veterinary Science Development ◽

10.4081/vsd.2013.4703 ◽

2013 ◽

Vol 3 (1) ◽

pp. 6 ◽

Cited By ~ 2

Author(s):

Ted H. Elsasser ◽

Stanislaw Kahl ◽

Katie M. Lebold ◽

Maret G. Traber ◽

Jessica Shaffer ◽

...

Keyword(s):

Plasma Concentrations ◽

Short Term ◽

Proinflammatory Response ◽

E Coli ◽

Amyloid A ◽

Proinflammatory Mediators ◽

Tnf Α ◽

Factor Α ◽

Necrosis Factor

While vitamin E has been used for decades in cattle diets, the principle form used traditionally is the synthetic α-isoform acetate or succinate and largely no data exist on the biological partitioning or functionality of the major naturally occurring γ- and δ-isoforms in cattle. Using tyrosine 3’-nitrated protein (pNT) as a biomarker of nitrosative cell stress, we sought to evaluate the effectiveness of short-term feeding supplementation of high content natural α-tocopherol (α-T, 96% α-isomer) compared to high content γ- and δ-enriched low α-content mixed tocopherol oils (γ-T, ~70% γ-, 20% δ-, <5% α-isoform) to mitigate systemic and hepatic aspects of the proinflammatory response to endotoxin (LPS). Calves fed diets supplemented with α-T, γ-T for five days or no tocopherol supplement (T0E) were challenged with a low-level of LPS (0.25 μg/kg, iv, E. coli 055:B5) sufficient to effect a liver nitration response. As fed, α-T or γ-T increased plasma and liver content of the respective tocopherols reflecting their relative abundance in the respective diets. Plasma or tissue mediators and biomarkers of the proinflammatory response [plasma concentrations of tumor necrosis factor-α (TNF-α, P<0.001), nitrate+nitrite (NOx, P<0.01), and serum amyloid A (SAA, P<0.001)], and general liver content of pNT (P<0.005) increased after LPS. LPS-mediated increases in TNF-α were not dif- ferent between diet treatments; both plasma NOx (P<0.05) and generalized liver pNT (P<0.03) responses were attenuated significantly in α-T and γ-T versus T0E calves. Plasma SAA was significantly decreased in γ-T calves at 24 h post-LPS relative to responses in α-T or T0E calves. The nitration of the mitochondrial proteins 24 h post-LPS was not only attenuated in α-T and γ-T vs T0E, but also the mitigating effect of γ-T on these specific nitration events was greater than that of α-T (P<0.01). Results are consistent with the concept that short-term α-T or γ-T supplementation can effectively decrease proinflammatory liver pNT after LPS; some mitochondrial nitration targets may be better protected with prophylactic supplementation with γ-,δ-tocopherol enriched oil.

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Effect of sample type on plasma concentrations of cell-free DNA and nucleosomes in dogs

Veterinary Record Open ◽

10.1136/vetreco-2019-000357 ◽

2019 ◽

Vol 6 (1) ◽

pp. e000357 ◽

Cited By ~ 2

Author(s):

Robert Goggs

Keyword(s):

Plasma Concentrations ◽

Correlation Coefficients ◽

Neutrophil Extracellular Trap ◽

Sample Type ◽

Cell Free Dna ◽

Free Dna ◽

Immune Mediated ◽

Deming Regression ◽

Significant Difference ◽

Highly Correlated

Cell-free DNA (cfDNA) and nucleosomes are two biomarkers of cell death and neutrophil extracellular trap formation that are increased in dogs with sepsis, immune-mediated haemolytic anaemia, cancer and following trauma and have diagnostic and prognostic values. cfDNA and nucleosomes are typically measured in plasma samples using DNA-specific fluorophores and ELISA assays, respectively, but their concentrations may be affected by pre-analytical variables such as sample type. The present study aimed to investigate the influence of sample type on the plasma cfDNA and nucleosome concentrations of a heterogeneous group of dogs presenting to an emergency room. Triplicate samples were collected into K2-ethylenediamine tetraacetic acid, 3.2% citrate and a specialised DNA stabilisation tube (Streck BCT), processed rapidly and frozen for batch analysis. Biomarker concentrations were compared between sample types by calculation of Spearman’s correlation coefficients, and with Deming regression, Bland-Altman plots and the Friedman test. Overall, biomarker concentrations were highly correlated between the three sample types. The most concordant results were obtained using citrate samples and the DNA stabilisation tube. Matched cfDNA concentrations between the different sample types were significantly different but there was no significant difference between the nucleosome concentrations in any of the sample types. The present study suggests that cfDNA and nucleosomes can be successfully measured in various sample types, but distinct sample types do not produce interchangeable results. This argues for use of a consistent sample type within studies and suggests standardisation may be useful for the field.

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Tumor cell-free DNA copy number instability compared to CA19-9 as an early predictor of response to systemic therapy in pancreatic ductal adenocarcinoma (PDAC).

Journal of Clinical Oncology ◽

10.1200/jco.2017.35.15_suppl.e14524 ◽

2017 ◽

Vol 35 (15_suppl) ◽

pp. e14524-e14524

Author(s):

Madappa N. Kundranda ◽

Alexander Koenig ◽

Julia Beck ◽

Kirsten Bornemann-Kolatzki ◽

Jessica Coats ◽

...

Keyword(s):

Pancreatic Ductal Adenocarcinoma ◽

Real Time ◽

Systemic Therapy ◽

Copy Number ◽

Ductal Adenocarcinoma ◽

Malignant Neoplasms ◽

Cell Free Dna ◽

Free Dna ◽

Time Assessment

e14524 Background: Humoral tumor markers are used clinically for real-time assessment of therapeutic efficacy. In pancreatic ductal adenocarcinoma (PDAC) the predominant marker is CA19-9, which is not expressed by 10 to 30% of patients depending on race. We compared plasma cell-free DNA (cfDNA) copy number based assay with changes in serum CA19-9 levels and radiological responses to predict responses to systemic therapy. Methods: In a laboratory blinded, prospective multicenter pilot study, 40 non-resectable PDAC patients, treated with (m)FOLFIRINOX, CAPIRI, or gemcitabine +/- nab-paclitaxel) are currently enrolled. CA19-9 was determined in the local center’s laboratory. Tumor cfDNA was measured with a copy-number instability (CNI) scoring assay, determined by next generation sequencing in a centralized laboratory. The CNI score assesses the amount of cfDNA with somatic macro-alterations originating from malignant neoplasms. The difference of the values before commencing therapy (baseline) and prior to cycle 2 (either rising or falling) was calculated as a predictor of standardized radiological evaluation of chemotherapeutic efficacy. Results: 37 patients (3 drop-outs) had data for baseline and cycle 2, of which CA19-9 was elevated and evaluable in 29 patients. The direction from baseline to cycle 2 of CA19-9 and CNI scores were in agreement in 18/29 patients. 9 of 11 cases with discordant CNI score and CA19-9 had treatment response data, and CNI correlated with 7/9 (78%); in contrast 7/9 had rising CA19-9, when response was stable disease or better (22% concordance). In the 27 patients with available imaging, CNI predicted better (n = 18) than CA19-9 (n = 10) (p = 0.03 Fisher’s exact). Conclusions: This comparative study on cfDNA versus CA19-9 suggest that cfDNA CNI quantitation is a potentially more reliable blood based marker for early real-time assessment of efficacy in systemic PDAC therapy than CA19-9, compared to standard of care imaging. The better prediction after the first cycle might be due to the very short in vivo half-life of cfDNA ( < 1hr) compared to about one week for CA19-9. These results justify a larger prospective validation trial.

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Factors regulating the rat insulin-like growth factor-binding protein-1 response to octreotide

Journal of Endocrinology ◽

10.1677/joe.0.1580061 ◽

1998 ◽

Vol 158 (1) ◽

pp. 61-68

Author(s):

MS Lewitt ◽

D Cameron-Smith ◽

NM Clarke ◽

H Saunders ◽

JL Phuyal

Keyword(s):

Growth Factor ◽

Time Course ◽

Binding Protein ◽

Similar Response ◽

Insulin Like Growth Factor ◽

High Fat ◽

Short Term ◽

Factor Binding ◽

The Mean

Insulin-like growth factor-binding protein-1 (IGFBP-1) production is increased by somatostatin and its analogues. In order to determine the time course and identify possible mechanisms of this increase in vivo we administered octreotide to rats and determined IGFBP-1 concentrations by RIA. After 60 min of anaesthesia, the mean baseline IGFBP-1 concentrations were 166 (95% confidence interval 123 to 225) ng/ml and increased in saline-infused animals to 729 (488 to 1086) ng/ml after 180 min. IGFBP-1 was stimulated transiently in response to octreotide, with circulating IGFBP-1 concentrations peaking at 1605 (1220 to 2111) ng/ml at 105 min during a continuous infusion of octreotide (100 micrograms/kg per h). In conscious chronically cannulated rats, baseline IGFBP-1 concentrations were 22 (18 to 28) ng/ml, 8-fold less than in the anaesthetised state, and were stimulated in the short term after administration of an octreotide bolus (100 micrograms/kg s.c.) to reach 88 (62 to 126) ng/ml at 60 min. A similar response was seen after i.v. administration to conscious rats. Intravenous bolus of octreotide (100 micrograms/kg) in rats anaesthetised for 3 h resulted in an increase in IGFBP-1 to peak at 1556 (1268 to 1910) ng/ml at 60 min. The IGFBP-1 response to octreotide was diminished in high-fat fed hyperinsulinaemic rats. The pattern of disappearance of iodinated IGFBP-1 from the circulation was not influenced by octreotide. The changes in GH, insulin and glucose concentrations alone did not sufficiently account for the patterns of response observed. We conclude that, in rats, octreotide stimulates IGFBP-1 acutely and this response is potentiated by factors related to anaesthesia.

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